Analysis of transcription in lens using tiled microarrays (ChIP on chip)

描述（由申请人提供）：该R21申请的长期目标是使用染色质免疫沉淀（chlp）对晶状体中的基因表达进行综合探索性研究，然后使用Nimblegen开发的高密度平铺寡核苷酸阵列（芯片上的chlp）进行分析。该方法将用于两种模式生物（小鼠和鸡）脊椎动物晶状体发育过程中的数据收集、跨物种比较和调控网络的解剖。该提案将(1)确定晶状体发育和体内平衡必需蛋白的近100个基因/位点的晶状体谱系形成、Pax6和Sox2以及分化的5个关键转录因子（c-Maf、Proxl和pRb）的结合位点；(2)鉴定这些基因/位点的染色质结构，重点关注组蛋白H3 K9乙酰化、H3 K3/27甲基化、DMA甲基化、绝缘子蛋白CTCF和染色质激活剂HMGN3的结合，这些基因/位点都参与表观遗传调控。这些相互作用将在小鼠和鸡晶体中确定，每个位点在10到500 kb之间，平铺在基于定制的微阵列上。这些基因座将包括所有晶状体结构基因（晶体蛋白、膜蛋白和中间丝蛋白）、调节晶状体谱系形成的关键基因（如Pax6、Six3和Sox2）、其末端分化（Proxl、c-Maf和Sox1）和晶状体发育过程中活跃的信号转导网络组分（如FGFR1至4）。这些数据将与RNA表达研究相关联，选定的数据将通过分子生物学实验进行验证。该数据将为鉴定体内单个基因座远端调控区域的功能、鉴定各种转录因子之间的交叉调控以及系统分析功能丧失和功能获得模型中的晶状体发育奠定基础。这些数据的生成和分析将用于新的聚焦R01项目，以阐明先天性晶状体缺陷和白内障中正常晶状体发育和异常基因调控。

DESCRIPTION (provided by applicant): The long-term goal of this R21 application is to perform integrative exploratory studies of gene expression in lens using chromatin immunoprecipitations (ChlPs) followed by analysis using high-density tiled oligonucleotide arrays (chips) developed by Nimblegen (ChlPs on chip). This approach will be used for massive acceleration of data collection, cross-species comparisons and dissection of regulatory networks during vertebrate lens development in 2 model organisms, mouse and chicken. This proposal will (1) Determine the profiles of binding sites of 5 key transcription factors regulating lens lineage formation, Pax6 and Sox2, and differentiation (c-Maf, Proxl and pRb) with nearly 100 genes/loci encoding proteins essential for lens development and homeostasis; and (2) Identify chromatin structure of these genes/loci focusing on histone H3 K9 acetylation, H3 K3/27 methylation, methylation of DMA, binding of insulator protein CTCF and chromatin activator HMGN3, all involved in epigenetic regulation. These interactions will be determined in mouse and chicken lens with each locus between 10 to 500 kb, tiled on custom-based microarrays. These loci will include all lens structural genes (crystallins, membrane and intermediate filament proteins), key genes regulating lens lineage formation (e.g., Pax6, Six3 and Sox2), its terminal differentiation (Proxl, c-Maf and Sox1) and components of signal transduction networks (e.g., FGFR1 to 4) active during lens development. The data will be correlated with RNA expression studies and selected data will be validated using molecular biology experiments. This data will lay a foundation for the functional identification of distal regulatory regions of individual loci in vivo, identification of cross-regulation between various transcription factors, and systemic analysis of lens development in loss-of-and gain-of-function models. Generation and analysis of this data will be used for new focused R01 projects to elucidate normal lens development and abnormal gene regulation during congenital lens defects and in cataracts.